Mandelic acid derivatives and their use as thrombin inhibitors

ABSTRACT

There is provided a compound of formula (1) and pharmaceutically-acceptable derivatives (including prodrugs) thereof. Which compound and derivatives are useful as, or are as useful as prodrugs of, competitive inhibitors of trypsin-like proteases, such as thrombin, and thus in particular, in the treatment of conditions where inhibition of thrombin is required (e.g. thrombosis) or as anticoagulants.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application PCT/SE02/01557, filed Aug. 30, 2002, whichclaims priority from Sweden Application No. 0102921-4, filed Aug. 30,2001, the specifications of each of which are incorporated by referenceherein. International Application PCT/SE02/01557 was published under PCTArticle 21(2) in English.

FIELD OF THE INVENTION

This invention relates to novel pharmaceutically useful compounds, inparticular compounds that are, and/or compounds that are metabolised tocompounds which are, competitive inhibitors of trypsin-like serineproteases, especially thrombin, their use as medicaments, pharmaceuticalcompositions containing them and synthetic routes to their production.

BACKGROUND

Blood coagulation is the key process involved in both haemostasis (i.e.the prevention of blood loss from a damaged vessel) and thrombosis (i.e.the formation of a blood clot in a blood vessel, sometimes leading tovessel obstruction).

Coagulation is the result of a complex series of enzymatic reactions.One of the ultimate steps in this series of reactions is the conversionof the proenzyme prothrombin to the active enzyme thrombin.

Thrombin is known to play a central role in coagulation. It activatesplatelets, leading to platelet aggregation, converts fibrinogen intofibrin monomers, which polymerise spontaneously into fibrin polymers,and activates factor XIII, which in turn crosslinks the polymers to forminsoluble fibrin. Furthermore, thrombin activates factor V and factorVIII leading to a “positive feedback” generation of thrombin fromprothrombin.

By inhibiting the aggregation of platelets and the formation andcrosslinking of fibrin, effective inhibitors of thrombin would beexpected to exhibit antithrombotic activity. In addition, antithromboticactivity would be expected to be enhanced by effective inhibition of thepositive feedback mechanism.

PRIOR ART

The early development of low molecular weight inhibitors of thrombin hasbeen described by Claesson in Blood Coagul. Fibrinol. (1994) 5, 411.

Blombäck et al (in J. Clin. Lab. Invest. 24, suppl. 107, 59, (1969))reported thrombin inhibitors based on the amino acid sequence situatedaround the cleavage site for the fibrinogen Aα chain. Of the amino acidsequences discussed, these authors suggested the tripeptide sequencePhe-Val-Arg (P9-P2-P1, hereinafter referred to as the P3-P2-P1 sequence)would be the most effective inhibitor.

Thrombin inhibitors based on dipeptidyl derivatives with anα,ω-aminoalkyl guanidine in the P1-position are known from U.S. Pat. No.4,346,078 and International Patent Application WO 93/11152. Similar,structurally related, dipeptidyl derivatives have also been reported.For example International Patent Application WO 94/29336 disclosescompounds with, for example, aminomethyl benzamidines, cyclic aminoalkylamidines and cyclic aminoalkyl guanidines in the P1-position(International Patent Application WO 97/23499 discloses prodrugs ofcertain of these compounds); European Patent Application 0 648 780,discloses compounds with, for example, cyclic aminoalkyl guanidines inthe P1-position.

Thrombin inhibitors based on peptidyl derivatives, also having cyclicaminoalkyl guanidines (e.g. either 3- or4-aminomethyl-1-amidino-piperidine) in the P1-position are known fromEuropean Patent Applications 0 468 231, 0 559 046 and 0 641 779.

Thrombin inhibitors based on tripeptidyl derivatives with argininealdehyde in the P1-position were first disclosed in European PatentApplication 0 185 390.

More recently, arginine aldehyde-based peptidyl derivatives, modified inthe P3-position, have been reported. For example, International PatentApplication WO 93/18060 discloses hydroxy acids, European PatentApplication 0 526 877 des-amino acids, and European Patent Application 0542 525 O-methyl mandelic acids in the P3-position.

Inhibitors of serine proteases (e.g. thrombin) based on electrophilicketones in the P1-position are also known. For example, European PatentApplication 0 195 212 discloses peptidyl α-keto esters and amides,European Patent Application 0 362 002 fluoroalkylamide ketones, EuropeanPatent Application 0 364 344 α,β,δ-triketocompounds, and European PatentApplication 0 530 167 α-alkoxy ketone derivatives of arginine in theP1-position.

Other, structurally different, inhibitors of trypsin-like serineproteases based on C-terminal boronic acid derivatives of arginine andisothiouronium analogues thereof are known from European PatentApplication 0 293 881.

More recently, thrombin inhibitors based on peptidyl derivatives havebeen disclosed in European Patent Application 0 669 317 andInternational Patent Applications WO 95/35309, WO 95/23609, WO 96/25426,WO 97/02284, WO 97/46577, WO 96/32110, WO 96/31504, WO 96/03374, WO98/06740, WO 97/49404, WO 98/57932, WO 99/29664, WO 00/35869 and WO00/42059.

In particular, WO 97/02284 and WO 00/42059 disclose thrombin inhibitorswith substituted mandelic acids in the P3 position.

However, there remains a need for effective inhibitors of trypsin-likeserine proteases, such as thrombin. There is also a need for compoundswhich to have a favourable pharmacokinetic profile and are selective ininhibiting thrombin over other serine proteases, in particular thoseinvolved in haemostasis. Compounds which exhibit competitive inhibitoryactivity towards thrombin would be expected to be especially useful asanticoagulants and therefore in the therapeutic treatment of thrombosisand is related disorders.

DISCLOSURE OF THE INVENTION

According to the invention there is provided a compound of formula I

(i.e. Ph(3-Cl)(5-OCHF₂)—CH(OH)C(O)-Aze-Pab(2,6-diF)), or apharmaceutically-acceptable derivative thereof.

The term “pharmaceutically-acceptable derivative” includespharmaceutically-acceptable salts (e.g. acid addition salts).

Abbreviations are listed at the end of this specification.

The compound of formula I may be made in accordance with techniques wellknown to those skilled in the art, for example as described hereinafter.

According to a further aspect of the invention there is provided aprocess for the preparation of the compound of formula I, whichcomprises:

(i) the coupling of a compound of formula II,

with a compound of formula III,

for example in the presence of a coupling agent (e.g. oxalyl chloride inDMF, EDC, DCC, HBTU, HATU, PyBOP or TBTU), an appropriate base (e.g.pyridine, DMAP, TEA, 2,4,6-collidine or DIPEA) and a suitable organicsolvent (e.g. dichloromethane, acetonitrile, EtOAc or DMF);

(ii) the coupling of a compound of formula IV,

with the compound of formula V,

for example under conditions as described in process (i) above; or

(iii) reaction of a corresponding compound of formula XVI, as definedhereinafter, with a suitable source of ammonia (e.g. ammonium acetate orammonia gas) under conditions known to those skilled in the art, such asby reaction of an ethylimidoate intermediate (formed by reaction of acompound of formula XVI with HCl(g) in ethanol) with ammonia gas inethanol, or under those conditions described in Tetrahedron Lett. 40,7067 (1999), the disclosures in which document are hereby incorporatedby reference.

Compounds of formula II are available using known and/or standardtechniques.

For example, compounds of formula II may be prepared by reaction of thealdehyde of formula VI,

with:

(a) a compound of formula VII,R″CN  VIIwherein R″ represents H or (CH₃)₃Si, for example at room, or elevated,temperature (e.g. below 100° C.) in the presence of a suitable organicsolvent (e.g. chloroform or methylene chloride) and, if necessary, inthe presence of a suitable base (e.g. TEA) and/or a suitable catalystsystem (e.g. benzylammonium chloride or zinc iodide, or using a chiralcatalyst, for example as described in Chem. Rev., (1999) 99, 3649),followed by hydrolysis under conditions that are well known to thoseskilled in the art (e.g. as described hereinafter);

(b) NaCN or KCN, for example in the presence of NaHSO₃ and water,followed by hydrolysis;

(c) chloroform, for example at elevated temperature (e.g. above roomtemperature but below 100° C.) in the presence of a suitable organicsolvent (e.g. chloroform) and, if necessary, in the presence of asuitable catalyst system (e.g. benzylammonium chloride), followed byhydrolysis;

(d) a compound of formula VIII,

wherein M represents Mg or Li, followed by oxidative cleavage (e.g.ozonolysis or osmium or ruthenium catalysed) under conditions which arewell known to those skilled in the art; or

(e) tris(methylthio)methane under conditions which are well known tothose is skilled in the art, followed by hydrolysis in the presence ofe.g. HgO and HBF₄.

Compounds of formula II may alternatively be prepared by oxidation of acompound of formula IX,

or a derivative thereof that is optionally protected at the secondaryhydroxyl group, in the presence of a suitable oxidising agent (e.g. acombination of a suitable free radical oxidant (such as TEMPO) and anappropriate hypochlorite salt (such as sodium hypochlorite)) underconditions known to those skilled in the art, for example at between−10° C. and room temperature, in the presence of a suitable solvent(e.g. water, acetone or a mixture thereof), an appropriate salt (e.g. analkali metal halide such as potassium bromide) and a suitable base (e.g.an alkali metal carbonate or hydrogen carbonate such as sodium hydrogencarbonate).

Enantiomerically-pure forms of compounds of formula II (i.e. thosecompounds having different configurations of substituents about theC-atom α- to the CO₂H group) may be separated by an enantiospecificderivatisation step. This may be achieved, for example by an enzymaticprocess. Such enzymatic processes include, for example,transesterification of the α-OH group at between room and refluxtemperature (e.g. at between 45 and 65° C.) in the presence of asuitable enzyme (e.g. Lipase PS Amano), an appropriate ester (e.g. vinylacetate) and a suitable solvent (e.g. methyl tert-butyl ether). Thederivatised isomer may then be separated from the unreacted isomer byconventional separation techniques (e.g. chromatography).

Groups added to compounds of formula II in such a derivatisation stepmay be removed either before any further reactions or at any later stagein the synthesis of compounds of formula I. The additional groups may beremoved using conventional techniques (e.g. for esters of the α-OHgroup, hydrolysis under conditions known to those skilled in the art(e.g. at between room and reflux temperature in the presence of asuitable base (e.g. NaOH) and an appropriate solvent (e.g. MeOH, wateror mixtures thereof))).

Compounds of formula III may be prepared by couplingazetidine-2-carboxylic acid to a compound of formula V, as hereinbeforedefined, for example under similar conditions to those described hereinfor preparation of compounds of formula I.

Compounds of formula IV may be prepared by coupling a compound offormula II as hereinbefore defined to azetidine-2-carboxylic acid, forexample under similar conditions to those described herein forpreparation of compounds of formula I.

The compound of formula VI is available using known and/or standardtechniques. For example, it may be prepared by:

(i) metallation (wherein the metal may be, for example, an alkali metalsuch as Li or, preferably, a divalent metal such as Mg) of a compound offormula X,

wherein Hal represents a halogen atom selected from Cl, Br and I,followed by reaction with a suitable source of the formyl group (such asN,N-dimethylformamide), for example under conditions describedhereinafter;

(ii) reduction of a compound of formula XI,

in the presence of a suitable reducing agent (e.g. DIBAL-H); or

(iii) oxidation of a compound of formula XII,

in the presence of a suitable oxidising agent (e.g. MnO₂, pyridiniumchlorochromate, a combination of DMSO and oxalyl chloride, or SO₃pyridine complex in DMSO).

Compounds of formula IX may be prepared by dihydroxylation of acorresponding compound of formula XIII

in the presence of a suitable dihydroxylating agent (e.g. a reagent orreagent mixture that provides OsO₄, such as AD-mix-α or, particularly,AD-mix-β), for example under conditions known to those skilled in theart, such as at between −10° C. and room temperature in the presence ofan appropriate solvent (e.g. water, tert-butanol or a mixture thereof).When asymmetric oxidants such as AD-mix-α or AD-mix-β are employed, thismethod may be used to prepare compounds of formula IX that have specificconfigurations of groups (i.e. R or S) about both of the C-atoms towhich the primary and secondary hydroxyl groups are attached.

The compound of formula XIII may be prepared by reaction of acorresponding compound of formula X, as hereinbefore defined, with asuitable source of the vinyl anion (e.g. tributyl(vinyl)tin) underconditions known to those skilled in the art, for example at betweenroom and reflux temperature (e.g. 50° C.) in the presence of anappropriate solvent (e.g. toluene), a suitable coupling agent (e.g. apalladium(0) co-ordination complex such astetrakis(triphenylphosphine)palladium(0)) and optionally in the presenceof an appropriate catalyst (e.g. 2,6-di-tert-butyl-4-methylphenol).

Compounds of formulae V, VII, VIII, X, XI, XII andazetidine-2-carboxylic acid are either commercially available, are knownin the literature, or may be obtained either by analogy with theprocesses described herein, or by conventional synthetic procedures, inaccordance with standard techniques, from readily available startingmaterials using appropriate reagents and reaction conditions. Compoundsof formula XVI may be obtained by processes described hereinafter.

Substituents on the phenyl ring in compounds of formulae I, II, III, IV,V, VI, IX, X, XI, XII and XIII may be introduced using techniques wellknown to those skilled in the art by way of standard functional groupsinterconversions, in accordance with standard techniques, from readilyavailable starting materials using appropriate reagents and reactionconditions.

For example, compounds of formulae I, II, IV, VI, X, XI and XII may beprepared from compounds corresponding to those compounds, in which, inplace of the —OCHF₂ group, an —OH groups is present (hereinafterreferred to as “the relevant phenol precursor compounds”), for exampleby reaction of such a relevant phenol precursor compound with anappropriate fluorinated haloalkane (such as ClCHF₂), e.g. at roomtemperature or above (e.g. at reflux) in the presence of a suitable base(e.g. potassium tert-butoxide, KOH or NaOH, for example in aqueoussolution) and an appropriate organic solvent (e.g. THF, chloroform ori-propanol), for example as described hereinafter.

The skilled person will appreciate that such functional grouptransformations may also be carried out at an earlier stage in theoverall synthesis of compounds of formulae II, IV, VI, X, XI and XII(i.e. on appropriate precursors of the relevant phenol precursorcompounds). The relevant phenol precursor compounds are eithercommercially available, are known in the literature, or may be obtainedeither by analogy with the processes described herein, or byconventional synthetic procedures, in accordance with standardtechniques, from readily available starting materials using appropriatereagents and reaction conditions. For example, the relevant phenolprecursor compounds may be obtained by deprotection of the correspondingprotected phenols (where the protecting group may be, for example,methyl, allyl, benzyl or tert-butyl) under standard conditions.

Compounds of formula I may be isolated from their reaction mixturesusing conventional techniques.

In accordance with the present invention, pharmaceutically acceptablederivatives of compounds of formula I also include “protected”derivatives, and/or compounds that act as prodrugs, of compounds offormula I.

Compounds that may act as prodrugs of compounds of formula I that may bementioned include compounds of formula Ia,

wherein R¹ represents OR² or C(O)OR³;

-   R² represents H, C₁₋₁₀ alkyl, C₁₋₃ alkylaryl or C₁₋₃ alkyloxyaryl    (the alkyl parts of which latter two groups are optionally    interrupted by one or more oxygen atoms, and the aryl parts of which    latter two groups are optionally substituted by one or more    substituents selected from halo, phenyl, methyl or methoxy, which    latter three groups are also optionally substituted by one or more    halo substituents); and-   R³ represents C₁₋₁₀ alkyl (which latter group is optionally    interrupted by one or more oxygen atoms), or C₁₋₃ alkylaryl or C₁₋₃    alkyloxyaryl (the alkyl parts of which latter two groups are    optionally interrupted by one or more oxygen atoms, and the aryl    parts of which latter two groups are optionally substituted by one    or more substituents selected from halo, phenyl, methyl or methoxy,    which latter three groups are also optionally substituted by one or    more halo substituents), and pharmaceutically-acceptable derivatives    thereof.

The term “pharmaceutically-acceptable derivatives” of compounds offormula Ia includes pharmaceutically-acceptable salts (e.g. acidaddition salts).

Alkyloxyaryl groups that R² and R³ may represent comprise an alkyl andan aryl group linked by way of an oxygen atom. Alkylaryl andalkyloxyaryl groups are linked to the rest of the molecule via the alkylpart of those groups, which alkyl parts may (if there is a sufficientnumber (i.e. three) of carbon atoms) be branched-chain. The aryl partsof alkylaryl and alkyloxyaryl groups which R² and R³ may represent, orbe substituted by, include carbocyclic and heterocyclic aromatic groups,such as phenyl, naphthyl, pyridinyl, oxazolyl, isoxazolyl, thiadiazolyl,indolyl and benzofuranyl and the like.

Alkyl groups which R² and R³ may represent may be straight-chain or,when there is a sufficient number (i.e. a minimum of three) of carbonatoms, be branched-chain and/or cyclic. Further, when there is asufficient number (i.e. a minimum of four) of carbon atoms, such alkylgroups may also be part cyclic/acyclic. Such alkyl groups may also besaturated or, when there is a sufficient number (i.e. a minimum of two)of carbon atoms, be unsaturated.

Halo groups with which R² and R³ may be substituted include fluoro,chloro, bromo and iodo.

When R¹ represents C(O)OR³, preferred R³ groups include:

-   -   (a) linear, branched or cyclic C₃₋₆ alkyl, for example C₄₋₆        cycloalkyl;    -   (b) C₁₋₂ alkylaryl groups, such as benzyl, optionally        substituted as indicated hereinbefore.

Preferred compounds of formula Ia include those in which R¹ representsOR².

When R¹ represents OR², preferred R² groups include:

-   -   (a) H;    -   (b) unsubstituted, linear, branched or cyclic C₁₋₈ (e.g. C₁₋₆)        alkyl, such as linear C₁₋₃ alkyl (e.g. ethyl or, particularly,        methyl), branched C₃₋₈ alkyl (e.g. i-propyl, i-butyl or        4-heptyl) or cyclic C₄₋₇ alkyl (i.e. C₄₋₇ cycloalkyl, e.g.        cyclobutyl or cyclohexyl);    -   (c) C₁₋₃ alkyloxyphenyl (e.g. C₂ alkyloxyphenyl), which phenyl        group is optionally substituted by one or more substituents as        indicated hereinbefore (e.g. trifluoromethyl);    -   (d) C₁₋₂ alkylaryl (e.g. methylaryl), wherein the aryl group is        phenyl, pyridinyl, oxazolyl or isoxazolyl, which latter three        groups are optionally substituted by one or more substituents as        indicated hereinbefore (e.g. methoxy, methyl, bromo and/or        chloro).

Preferred compounds of formula Ia include those in which R¹ representsOR² and R² represents linear, branched (as appropriate), or cyclic (asappropriate), C₁₋₆ (e.g. C₁₋₄) alkyl, such as methyl, ethyl, n-propyl,i-propyl or cyclobutyl.

Compounds of formula Ia may be prepared by one or more of the followingmethods:

(a) reaction of a corresponding compound of formula II as hereinbeforedefined with a compound of formula XIV,

wherein R¹ is as hereinbefore defined, for example under similarconditions to those described hereinbefore for synthesis of compounds offormula I;

(b) reaction of a corresponding compound of formula IV as hereinbeforedefined with a compound of formula XV,

wherein R¹ is as hereinbefore defined, for example under similarconditions to those described hereinbefore for synthesis of compounds offormula I;

(c) for compounds of formula Ia in which R¹ represents OH, reaction of acorresponding compound of formula XVI,

with hydroxylamine, for example under conditions known to those skilledin the art;

(d) for compounds of formula Ia in which R¹ represents OR², reaction ofa protected derivative of a corresponding compound of formula I whichis, for example, a compound of formula XVII,

wherein R^(a) represents, for example, —CH₂CH₂—Si(CH₃)₃ or benzyl, or atautomer thereof, with a compound of formula XVIII,R²ONH₂  XVIIIwherein R² is as hereinbefore defined, or an acid addition salt thereof,for example at between room and reflux temperature in the presence of anappropriate organic solvent (e.g. THF, CH₃CN, DMF or DMSO), followed byremoval of the —C(O)OR^(a) group under conditions known to those skilledin the art (e.g. by reacting with QF or TFA (e.g. as describedhereinafter));

(e) for compounds of formula Ia in which R¹ represents OH, reaction of acompound of formula XVII, as hereinbefore defined, in which R^(a)represents benzyl with hydroxylamine, or an acid addition salt thereof,for example under conditions that will be well known to those skilled inthe art;

(f) for compounds of formula Ia in which R¹ represents COOR³, reactionof a corresponding compound of formula I as hereinbefore defined with acompound of formula XIX,L¹COOR³  XIXwherein L¹ represents a suitable leaving group, such as halo ornitrophenyl (e.g. 4-nitrophenyl), and R³ is as hereinbefore defined, forexample at or around room temperature in the presence of suitable base(e.g. NaOH, for example in aqueous solution) and an appropriate organicsolvent (e.g. methylene chloride); or

(g) for compounds of formula Ia in which R¹ represents OCH₃ or OCH₂CH₃,reaction of a corresponding compound of formula Ia in which R¹represents OH with dimethylsulfate or diethylsulfate, respectively, forexample in the presence of a suitable base (e.g. an alkali metalhydroxide such as KOH (for example in aqueous solution at e.g. 50 wt.%)) and an appropriate catalyst (e.g. a quaternary ammonium halide suchas benzyltrimethylammonium chloride (for example in CH₂Cl₂ or THFsolution at e.g. 10 wt. %)).

Compounds of formula XVI may be prepared by reaction of a correspondingcompound of formula II, as hereinbefore defined, with a compound offormula XX,

for example under similar conditions to those described hereinbefore forsynthesis of compounds of formula I.

Compounds of formulae XVI may alternatively be prepared by reaction of acorresponding compound of formula IV, as hereinbefore defined, with acompound of formula XXI,

for example under similar conditions to those described hereinbefore forsynthesis of compounds of formula I.

Compounds of formula XVII may be prepared by reaction of a correspondingcompound of formula II, as hereinbefore defined, with a compound offormula XXII,

wherein R^(a) are as hereinbefore defined, for example under similarconditions to those described hereinbefore for synthesis of compounds offormula I.

Alternatively, compounds of formula XVII may be prepared by reaction ofa corresponding compound of formula I with a compound corresponding to acompound of formula XIX in which, in place of R³, the group R^(a) ispresent, in which R^(a) is as hereinbefore defined, for example underconditions described above in respect of the preparation of compounds offormula Ia.

Compounds of formulae XIV and XXII may be prepared by reaction ofazetidine-2-carboxylic acid with, respectively, a compound of formula XVas hereinbefore defined, or a compound of formula XXIII,

wherein R^(a) is as hereinbefore defined, for example under similarconditions to those described hereinbefore for synthesis of compounds offormula I.

Compounds of formula XV, XVIII, XIX, XX, XXI and XXIII are eithercommercially available, are known in the literature, or may be obtainedeither by analogy with the processes described herein, or byconventional synthetic procedures, in accordance with standardtechniques, from readily available starting materials using appropriatereagents and reaction conditions. For example, compounds of formula XXmay be prepared by reaction of a corresponding compound of formula XXIwith azetidine-2-carboxylic acid, for example under similar conditionsto those described hereinbefore.

Compounds of formulae I and Ia, as defined above, and derivatives ofeither, are referred to hereinafter as “the compounds of the invention”.

The compounds of the invention may exhibit tautomerism. All tautomericforms and mixtures thereof are included within the scope of theinvention. Particular tautomeric forms that may be mentioned includethose connected with the position of the double bond in the amidinefunctionality in a compound of formula Ia, and the position of thesubstituent R¹.

Compounds of the invention also contain two or more asymmetric carbonatoms and may therefore exhibit optical and/or diastereoisomerism.Diastereoisomers may be separated using conventional techniques, e.g.chromatography. The various stereoisomers may be isolated by separationof a racemic or other mixture of the compounds using conventional, e.g.HPLC techniques. Alternatively the desired optical isomers may be madeby reaction of the appropriate optically active starting materials underconditions which will not cause racemisation or epimerisation, or byderivatisation, for example with a homochiral acid followed byseparation of the diastereomeric derivatives by conventional means (e.g.HPLC, chromatography over silica). All stereoisomers are included withinthe scope of the invention.

Compounds of the invention in which the

fragment is in the S-configuration are preferred.

Preferred compounds of the invention also include those in which thestructural fragment

is in the R-configuration.

The wavy lines on the bonds in the above two fragments signify the bondpositions of the fragments.

Thus, preferred compounds of the invention include:

-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF);-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe); and-   Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH).

It will be appreciated by those skilled in the art that in the processesdescribed above and hereinafter the functional groups of intermediatecompounds may need to be protected by protecting groups.

Functional groups that it is desirable to protect include hydroxy, aminoand carboxylic acid. Suitable protecting groups for hydroxy includeoptionally substituted and/or unsaturated alkyl groups (e.g. methyl,allyl, benzyl or tert-butyl), trialkylsilyl or diarylalkylsilyl groups(e.g. t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl) andtetrahydropyranyl. Suitable protecting groups for carboxylic acidinclude C₁₋₆ alkyl or benzyl esters.

Suitable protecting groups for amino and amidino includet-butyloxycarbonyl, benzyloxycarbonyl or 2-trimethylsilylethoxycarbonyl(Teoc). Amidino nitrogens may also be protected by hydroxy or alkoxygroups, and may be either mono- or diprotected.

The protection and deprotection of functional groups may take placebefore or after coupling, or before or after any other reaction in theabove-mentioned schemes.

Protecting groups may be removed in accordance with techniques that arewell known to those skilled in the art and as described hereinafter.

Persons skilled in the art will appreciate that, in order to obtaincompounds of the invention in an alternative, and, on some occasions,more convenient, manner, the individual process steps mentionedhereinbefore may be performed in a different order, and/or theindividual reactions may be performed at a different stage in theoverall route (i.e. substituents may be added to and/or chemicaltransformations performed upon, different intermediates to thosementioned hereinbefore in conjunction with a particular reaction). Thismay negate, or render necessary, the need for protecting groups.

The type of chemistry involved will dictate the need, and type, ofprotecting groups as well as the sequence for accomplishing thesynthesis.

The use of protecting groups is fully described in “Protective Groups inOrganic Chemistry”, edited by J W F McOmie, Plenum Press (1973), and“Protective Groups in Organic Synthesis”, 3^(rd) edition, T. W. Greene &P. G. M. Wutz, Wiley-Interscience (1999).

Protected derivatives of compounds of the invention may be convertedchemically to compounds of the invention using standard deprotectiontechniques (e.g. hydrogenation). The skilled person will also appreciatethat certain compounds of formula Ia may also be referred to as being“protected derivatives” of compounds of formula I.

Medical and Pharmaceutical Use

Compounds of the invention may possess pharmacological activity as such.Compounds of the invention that may possess such activity include, butare not limited to, compounds of formula I.

However, other compounds of the invention (including compounds offormula Ia) may not possess such activity, but may be administeredparenterally or orally, and may thereafter be metabolised in the body toform compounds that are pharmacologically active (including, but notlimited to, corresponding compounds of formula I). Such compounds (whichalso includes compounds that may possess some pharmacological activity,but that activity is appreciably lower than that of the “active”compounds to which they are metabolised), may therefore be described as“prodrugs” of the active compounds.

Thus, the compounds of the invention are useful because they possesspharmacological activity, and/or are metabolised in the body followingoral or parenteral administration to form compounds which possesspharmacological activity. The compounds of the invention are thereforeindicated as pharmaceuticals.

According to a further aspect of the invention there is thus providedthe compounds of the invention for use as pharmaceuticals.

In particular, compounds of the invention are potent inhibitors ofthrombin either as such and/or (e.g. in the case of prodrugs), aremetabolised following administration to form potent inhibitors ofthrombin, for example as may be demonstrated in the tests describedbelow.

By “prodrug of a thrombin inhibitor”, we include compounds that form athrombin inhibitor, in an experimentally-detectable amount, and within apredetermined time (e.g. about 1 hour), following oral or parenteraladministration (see, for example, Test E below) or, alternatively,following incubation in the presence of liver microsomes (see, forexample, Test G below).

The compounds of the invention are thus expected to be useful in thoseconditions where inhibition of thrombin is required, and/or conditionswhere anticoagulant therapy is indicated, including the following:

The treatment and/or prophylaxis of thrombosis and hypercoagulability inblood and/or tissues of animals including man. It is known thathypercoagulability may lead to thrombo-embolic diseases. Conditionsassociated with hypercoagulability and thrombo-embolic diseases whichmay be mentioned include inherited or acquired activated protein Cresistance, such as the factor V-mutation (factor V Leiden), andinherited or acquired deficiencies in antithrombin III, protein C,protein S, heparin cofactor II. Other conditions known to be associatedwith hypercoagulability and thrombo-embolic disease include circulatingantiphospholipid antibodies (Lupus anticoagulant), homocysteinemi,heparin induced thrombocytopenia and defects in fibrinolysis, as well ascoagulation syndromes (e.g. disseminated intravascular coagulation(DIC)) and vascular injury in general (e.g. due to surgery).

The treatment of conditions where there is an undesirable excess ofthrombin without signs of hypercoagulability, for example inneurodegenerative diseases such as Alzheimer's disease.

Particular disease states which may be mentioned include the therapeuticand/or prophylactic treatment of venous thrombosis (e.g. DVT) andpulmonary embolism, arterial thrombosis (e.g. in myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis), and systemic embolism usually from the atrium during atrialfibrillation (e.g. non-valvular atrial fibrillation) or from the leftventricle after transmural myocardial infarction, or caused bycongestive heart failure; prophylaxis of re-occlusion (i.e. thrombosis)after thrombolysis, percutaneous trans-luminal angioplasty (PTA) andcoronary bypass operations; the prevention of re-thrombosis aftermicrosurgery and vascular surgery in general.

Further indications include the therapeutic and/or prophylactictreatment of disseminated intravascular coagulation caused by bacteria,multiple trauma, intoxication or any other mechanism; anticoagulanttreatment when blood is in contact with foreign surfaces in the bodysuch as vascular grafts, vascular stents, vascular catheters, mechanicaland biological prosthetic valves or any other medical device; andanticoagulant treatment when blood is in contact with medical devicesoutside the body such as during cardiovascular surgery using aheart-lung machine or in haemodialysis; the therapeutic and/orprophylactic treatment of idiopathic and adult respiratory distresssyndrome, pulmonary fibrosis following treatment with radiation orchemotherapy, septic shock, septicemia, inflammatory responses, whichinclude, but are not limited to, edema, acute or chronic atherosclerosissuch as coronary arterial disease and the formation of atheroscleroticplaques, cerebral arterial disease, cerebral infarction, cerebralthrombosis, cerebral embolism, peripheral arterial disease, ischaemia,angina (including unstable angina), reperfilsion damage, restenosisafter percutaneous trans-luminal angioplasty (PTA) and coronary arterybypass surgery.

Compounds of the invention that inhibit trypsin and/or thrombin may alsobe useful in the treatment of pancreatitis.

The compounds of the invention are thus indicated both in thetherapeutic and/or prophylactic treatment of these conditions.

According to a further aspect of the present invention, there isprovided a method of treatment of a condition where inhibition ofthrombin is required which method comprises administration of atherapeutically effective amount of a compound of the invention to aperson suffering from, or susceptible to, such a condition.

The compounds of the invention will normally be administered orally,intravenously, subcutaneously, buccally, rectally, dermally, nasally,tracheally, bronchially, by any other parenteral route or viainhalation, in the form of pharmaceutical preparations comprisingcompound of the invention in a pharmaceutically acceptable dosage form.

Depending upon the disorder and patient to be treated and the route ofadministration, the compositions may be administered at varying doses.

The compounds of the invention may also be combined and/orco-administered with any antithrombotic agent(s) with a differentmechanism of action, such as one or more of the following: theantiplatelet agents acetylsalicylic acid, ticlopidine and clopidogrel;thromboxane receptor and/or synthetase inhibitors; fibrinogen receptorantagonists; prostacyclin mimetics; phosphodiesterase inhibitors;ADP-receptor (P₂T) antagonists; and inhibitors of carboxypeptidase U(CPU).

The compounds of the invention may further be combined and/orco-administered with thrombolytics such as one or more of tissueplasminogen activator (natural, recombinant or modified), streptokinase,urokinase, prourokinase, anisoylated plasminogen-streptokinase activatorcomplex (APSAC), animal salivary gland plasminogen activators, and thelike, in the treatment of thrombotic diseases, in particular myocardialinfarction.

According to a further aspect of the invention there is provided apharmaceutical formulation including a compound of the invention, inadmixture with a pharmaceutically acceptable adjuvant, diluent orcarrier.

Suitable daily doses of the compounds of the invention in therapeutictreatment of humans are about 0.001–100 mg/kg body weight at peroraladministration and 0.001–50 mg/kg body weight at parenteraladministration, excluding the weight of any acid counter-ion.

For the avoidance of doubt, as used herein, the term “treatment”includes therapeutic and/or prophylactic treatment.

Compounds of the invention have the advantage that they may be moreefficacious, be less toxic, be longer acting, have a broader range ofactivity, be more potent, produce fewer side effects, be more easilyabsorbed, and/or have a better pharmacokinetic profile (e.g. higher oralbioavailability and/or lower clearance), than, and/or have other usefulpharmacological, physical, or chemical, properties over, compounds knownin the prior art.

Compounds of the invention may have the further advantage that they maybe administered less frequently than compounds known in the prior art.

Biological Tests

The following test procedures may be employed.

Test A

Determination of Thrombin Clotting Time (TT)

The inhibitor solution (25 μL) is incubated with plasma (25 μL) forthree minutes. Human thrombin (T 6769; Sigma Chem. Co or HematologicTechnologies) in buffer solution, pH 7.4 (25 μL, 4.0 NIH units/mL), isthen added and the clotting time measured in an automatic device (KC 10;Amelung).

The thrombin clotting time (TT) is expressed as absolute values(seconds) as well as the ratio of TT without inhibitor (TT₀) to TT withinhibitor (TT_(i)). The latter ratios (range 1–0) are plotted againstthe concentration of inhibitor (log transformed) and fitted to sigmoidaldose-response curves according to the equationy=a/[1+(x/IC ₅₀)^(s)]where: a=maximum range, i.e. 1; s=slope of the dose-response curve; andIC₅₀=the concentration of inhibitor that doubles the clotting time. Thecalculations are processed on a PC using the software program GraFitVersion 3, setting equation equal to: Start at 0, define end=1(Erithacus Software, Robin Leatherbarrow,.Imperial College of Science,London, UK).Test BDetermination of Thrombin Inhibition with a Chromogenic, Robotic Assay

The thrombin inhibitor potency is measured with a chromogenic substratemethod, in a Plato 3300 robotic microplate processor (Rosys AG, CH-8634Hombrechtikon, Switzerland), using 96-well, half volume microtiterplates Costar, Cambridge, Mass., USA; Cat No 3690). Stock solutions oftest substance in DMSO (72 μL), 0.1–1 mmol/L, are diluted serially 1:3(24+48 μL) with DMSO to obtain ten different concentrations, which areanalysed as samples in the assay. 2 μL of test sample is diluted with124 μL assay buffer, 12 μL of chromogenic substrate solution (S-2366,Chromogenix, Mölndal, Sweden) in assay buffer and finally 12 μL ofα-thrombin solution (Human α-thrombin, Sigma Chemical Co. or HematologicTechnologies) in assay buffer, are added, and the samples mixed. Thefinal assay concentrations are: test substance 0.00068–13.3 μmol/L,S-2366 0.30 mmol/L, α-thrombin 0.020 NIHU/mL. The linear absorbanceincrement during 40 minutes incubation at 37° C. is used for calculationof percentage inhibition for the test samples, as compared to blankswithout inhibitor. The IC₅₀-robotic value, corresponding to theinhibitor concentration which causes 50% inhibition of the thrombinactivity, is calculated from a log concentration vs. % inhibition curve.

Test C

Determination of the Inhibition Constant K_(i) for Human Thrombin

K_(i)-determinations are made using a chromogenic substrate method,performed at 37° C. on a Cobas Bio centrifugal analyser (Roche, Basel,Switzerland). Residual enzyme activity after incubation of humanα-thrombin with various concentrations of test compound is determined atthree different substrate concentrations, and is measured as the changein optical absorbance at 405 nm.

Test compound solutions (100 μL; normally in buffer or saline containingBSA 10 g/L) are mixed with 200 μL of human α-thrombin (Sigma ChemicalCo) in assay buffer (0.05 mol/L Tris-HCl pH 7.4, ionic strength 0.15adjusted with NaCl) containing BSA (10 g/L), and analysed as samples inthe Cobas Bio. A 60 μL sample, together with 20 μL of water, is added to320 μL of the substrate S-2238 (Chromogenix AB, Mölndal, Sweden) inassay buffer, and the absorbance change (ΔA/min) is monitored. The finalconcentrations of S-2238 are 16, 24 and 50 μmol/L and of thrombin 0.125NIH U/mL.

The steady state reaction rate is used to construct Dixon plots, i.e.diagrams of inhibitor concentration vs. 1/(ΔA/min). For reversible,competitive inhibitors, the data points for the different substrateconcentrations typically form straight lines which intercept atx=−K_(i).

Test D

Determination of Activated Partial Thromboplastin Time (APTT)

APTT is determined in pooled normal human citrated plasma with thereagent PTT Automated 5 manufactured by Stago. The inhibitors are addedto the plasma (10 μL inhibitor solution to 90 μL plasma) and incubatedwith the APTT reagent for 3 minutes followed by the addition of 100 μLof calcium chloride solution (0.025 M) and APTT is determined by use ofthe coagulation analyser KC10 (Amelung) according to the instructions ofthe reagent producer.

The clotting time is expressed as absolute values (seconds) as well asthe ratio of APTT without inhibitor (APTT₀) to APTT with inhibitor(APTT_(i)). The latter ratios (range 1–0) are plotted against theconcentration of inhibitor (log transformed) and fitted to sigmoidaldose-response curves according to the equationy=a/[1+(x/IC ₅₀)^(s)]where: a=maximum range, i.e. 1; s=slope of the dose-response curve; andIC₅₀=the concentration of inhibitor that doubles the clotting time. Thecalculations are processed on a PC using the software program GraFitVersion 3, setting equation equal to: Start at 0, define end=1(Erithacus Software, Robin Leatherbarrow, Imperial College of Science,London, UK). IC₅₀APTT is defined as the concentration of inhibitor inhuman plasma that doubles the Activated Partial Thromboplastin Time.Test EDetermination of Thrombin Time ex vivo

The inhibition of thrombin after oral or parenteral administration ofthe compounds of the invention, dissolved in ethanol:SolutolK:water(5:5:90), is examined in conscious rats which, one or two days prior tothe experiment, are equipped with a catheter for blood sampling from thecarotid artery. On the experimental day blood samples are withdrawn atfixed times after the administration of the compound into plastic tubescontaining 1 part sodium citrate solution (0.13 mol per L) and 9 partsof blood. The tubes are centrifuged to obtain platelet poor plasma.

50 μL of plasma samples are precipitated with 100 μL of coldacetonitrile. The samples are centrifuged for 10 minutes at 4000 rpm. 75μL of the supernatant is diluted with 75 μL of 0.2% formic acid. 10 μLvolumes of the resulting solutions are analysed by LC-MS/MS and theconcentrations of thrombin inhibitor are determined using standardcurves.

Test F

Determination of Plasma Clearance in Rat

Plasma clearance is estimated in male Sprague Dawley rats. The compoundis dissolved in water and administered as a subcutaneous bolus injectionat a dose of 4 μmol/kg. Blood samples are collected at frequentintervals up to 5 hours after drug administration. Blood samples arecentrifuged and plasma is separated from the blood cells and transferredto vials containing citrate (10% final concentration). 50 μL of plasmasamples are precipitated with 100 μL of cold acetonitrile. The samplesare centrifuged for 10 minutes at 4000 rpm. 75 μL of the supernatant isdiluted with 75 μL of 0.2% formic acid. 10 μL volumes of the resultingsolutions are analysed by LC-MS/MS and the concentrations of thrombininhibitor are determined using standard curves. The area under theplasma concentration-time profile is estimated using the log/lineartrapezoidal rule and extrapolated to infinite time. Plasma clearance(CL) of the compound is then determined asCL=Dose/AUCThe values are reported in mL/min/kg.Test GDetermination of in vitro Stability

Liver microsomes are prepared from Sprague-Dawley rats and human liversamples according to internal SOPs. The compounds are incubated at 37°C. at a total microsome protein concentration of 3 mg/mL in a 0.05 mol/LTRIS buffer at pH 7.4, in the presence of the cofactors NADH (2.5mmol/L) and NADPH (0.8 mmol/L). The initial concentration of compound is5 or 10 μmol/L. Samples are taken for analysis up to 60 minutes afterthe start of the incubation. The enzymatic activity in the collectedsample is immediately stopped by adding 20% myristic acid at a volumecorresponding to 3.3% of the total sample volume. The concentration ofcompound remaining (FINAL CONC) in the 60 min. sample is determined bymeans of LCMS using a sample collected at zero time as reference (STARTCONC). The % of degraded thrombin inhibitor is calculated as:

$100\mspace{11mu}\% \times \frac{\left\lbrack {{START}\mspace{11mu}{CONC}} \right\rbrack - \left\lbrack {{FINAL}\mspace{14mu}{CONC}} \right\rbrack}{\left\lbrack {{START}\mspace{11mu}{CONC}} \right\rbrack}$Test HArterial Thrombosis Model

Vessel damage is induced by applying ferric chloride (FeCl₃) topicallyto the carotid artery. Rats are anaesthetised with an intraperitonealinjection of sodium pentobarbital (80 mg/kg; Apoteksbolaget; Ume{dotover (a)}, Sweden), followed by continuous infusion (12 mg/kg/h)throughout the experiment. Rat body temperature is maintained at 38° C.throughout the experiment by external heating. The experiment startswith a 5 minutes control period. Five minutes later, human¹²⁵I-fibrinogen (80 kBq; IM53; Amersham International, Buckinghamshire,UK) is given intravenously and is used as a marker for the subsequentincorporation of fibrin(ogen) into the thrombus. The proximal end of thecarotid artery segment is placed in a plastic tube (6 mm; Silastic®; DowCorning, Mich., USA) opened lengthways, containing FeCl₃-soaked (2 μL;55% w/w; Merck, Darmstadt, Germany) filter paper (diameter 3 mm; 1F;Munktell, Grycksbo, Sweden). The left carotid artery is exposed to FeCl₃for 10 minutes and is then removed from the plastic tube and soaked insaline. Fifty minutes later, the carotid artery is removed and rinsed insaline. Reference blood samples are also taken for determination ofblood ¹²⁵I-activity, 10 minutes after the injection of ¹²⁵I-fibrinogen,and at the end of the experiment. The ¹²⁵I-activity in the referenceblood samples and the vessel segment are measured in a gamma counter(1282 Compugamma; LKB Wallac Oy, Turku, Finland) on the same day as theexperiment is performed. The thrombus size is determined as the amountof ¹²⁵I-activity incorporated in the vessel segment in relation to the¹²⁵I-activity in the blood (cpm/mg).

General Experimental Details

TLC was performed on silica gel. Chiral HPLC analysis was performedusing a 46 mm×250 mm Chiralcel OD column with a 5 cm guard column.

The column temperature was maintained at 35° C. A flow rate of 1.0mL/min was used. A Gilson 115 UV detector at 228 nm was used. The mobilephase consisted of hexanes, ethanol and trifluroacetic acid and theappropriate ratios are listed for each compound. Typically, the productwas dissolved in a minimal amount of ethanol and this was diluted withthe mobile phase.

LC-MS/MS was performed using a HP-1100 instrument equipped with aCTC-PAL injector and a 5 μm, 4×100 mm ThermoQuest, Hypersil BDS-C18column. An API-3000 (Sciex) MS detector was used. The flow rate was 1.2mL/min and the mobile phase (gradient) consisted of 10–90% acetonitrilewith 90–10% of 4 mM aq. ammonium acetate, both containing 0.2% formicacid.

¹H NMR spectra were recorded using tetramethylsilane as the internalstandard. ¹³C NMR spectra were recorded using the listed deuteratedsolvents as the internal standard.

EXAMPLE 1 Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)

(i) 3-Chloro-5-methoxybenzaldehyde

3,5-Dichloroanisole (74.0 g, 419 mmol) in THF (200 mL) was addeddropwise to magnesium metal (14.2 g, 585 mmol, pre-washed with 0.5 NHCl) in THF (100 mL) at 25° C. After the addition, 1,2-dibromoethane(3.9 g, 20.8 mmol) was added dropwise. The resultant dark brown mixturewas heated at reflux for 3 h. The mixture was cooled to 0° C., andN,N-dimethylformamide (60 mL) was added in one portion. The mixture waspartitioned with diethyl ether (3×400 mL) and 6N HCl (500 mL). Thecombined organic extracts were washed with brine (300 mL), dried(Na₂SO₄), filtered and concentrated in vacuo to give an oil. Flashchromatography (2×) on silica get eluting with Hex:EtOAc (4:1) affordedthe sub-title compound (38.9 g, 54%) as a yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 9.90 (s, 1H), 7.53 (s, 1H), 7.38 (s, 1H), 7.15(s, 1H), 3.87 (s, 3H).

(ii) 3-Chloro-5-hydroxybenzaldehyde

A solution of 3-chloro-5-methoxybenzaldehyde (22.8 g, 134 mmol; see step(i) above) in CH₂Cl₂ (250 mL) was cooled to 0° C. Boron tribromide (15.8mL, 167 mmol) was added dropwise over 15 min. After stirring, thereaction mixture for 2 h, H₂O (50 mL) was added slowly. The solution wasthen extracted with Et₂O (2×100 mL). The organic layers were combined,dried (Na₂SO₄), filtered and concentrated in vacuo. Flash chromatographyon silica gel eluting with Hex:EtOAc (4:1) afforded the sub-titlecompound (5.2 g, 25%).

¹H NMR (300 MHz, CDCl₃) δ 9.85 (s, 1H), 7.35 (s, 1H), 7.20 (s, 1H), 7.10(s, 1H), 3.68 (s, 1H)

(iii) 3-Chloro-5-difluoromethoxybenzaldehyde

A solution of 3-chloro-5-hydroxybenzaldehyde (7.5 g, 48 mmol; see step(ii) above) in 2-propanol (250 mL) and 30% KOH (100 mL) was heated toreflux. While stirring, CHClF₂ was bubbled into the reaction mixture for2 h. The reaction mixture was cooled, acidified with 1N HCl andextracted with EtOAc (2×100 mL). The organics were washed with brine(100 mL), dried (Na₂SO₄), filtered and concentrated in vacuo. Flashchromatography on silica gel eluting with Hex:EtOAc (4:1) afforded thesub-title compound (4.6 g, 46%).

¹H NMR (300 MHz, CDCl₃) δ 9.95 (s, 1H), 7.72 (s, 1H), 7.52 (s, 1H), 7.40(s, 1H), 6.60 (t, J_(H-F)=71.1 Hz, 1H)

(iv) Ph(3-Cl)(5-OCHF₂)-(R,S)CH(OTMS)CN

A solution of 3-chloro-5-difluoromethoxybenzaldehyde (4.6 g, 22.3 mmol;see step (iii) above) in CH₂Cl₂ (200 mL) was cooled to 0° C. ZnI₂ (1.8g, 5.6 mmol) and trimethylsilyl cyanide (2.8 g, 27.9 mmol) were addedand the reaction mixture was allowed to warm to room temperature andstirred for 15 h. The mixture was partially concentrated in vacuoyielding the sub-title compound as a liquid, which was used directly instep (v) below without further purification or characterization.

(v) Ph(3-Cl)(5-OCHF₂)-(R,S)CH(OH)CH)OEt

Ph(3-Cl)(5-OCHF₂)-(R,S)CH(OTMS)CN (6.82 g, assume 22.3 mmol; see step(iv) above) was added dropwise to HCl/EtOH (500 mL). The reactionmixture was stirred 15 h, then partially concentrated in vacuo yieldingthe sub-title compound as a liquid, which was used in step (vi) withoutfurther purification or characterization.

(vi) Ph(3-Cl)(5-OCHF₂)-(R,S)CH(OH)C(O)OEt

Ph(3-Cl)(5-OCHF₂)-(R,S)CH(OH)C(NH)OEt (6.24 g, assume 22.3 mmol; seestep (v) above) was dissolved in THF (250 mL), 0.5M H₂SO₄ (400 mL) wasadded and the reaction was stirred at 40° C. for 65 h, cooled and thenpartially concentrated in vacuo to remove most of the THF. The reactionmixture was then extracted with Et₂O (3×100 mL), dried (Na₂SO₄),filtered and concentrated in vacuo to afford the sub-title compound as asolid, which was used in step (vii) without further purification orcharacterization.

(vii) Ph(3-Cl)(5-OCHF₂)-(R,S)CH(OH)C(O)OH

A solution of Ph(3-Cl)(5-OCHF₂)-(R,S)CH(OH)C(O)OEt (6.25 g, assume 22.3mmol; see step (vi) above) in 2-propanol (175 mL) and 20% KOH (350 mL)was stirred at room temperature 15 h. The reaction was then partiallyconcentrated in vacuo to remove most of the 2-propanol. The remainingmixture was acidified with 1M H₂SO₄, extracted with Et₂O (3×100 mL),dried (Na₂SO₄) and concentrated in vacuo to give a solid. Flashchromatography on silica gel eluting with CHCl₃:MeOH:concentrated NH₄OH(6:3:1) afforded the ammonium salt of the sub-title compound. Theammonium salt was then dissolved in a mixture of EtOAc (75 mL) and H₂O(75 mL) and acidified with 2N HCl. The organic layer was separated andwashed with brine (50 mL), dried (Na₂SO₄) and concentrated in vacuo toafford the sub-title compound (3.2 g, 57% from steps (iv) to (vii)).

¹H NMR (300 MHz, CD₃OD) δ 7.38 (s, 1H), 7.22 (s, 1H), 7.15 (s, 1H), 6.89(t, J_(H-F)=71.1 Hz, 1H), 5.16 (s, 1H)

(viii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)OH (a) andPh(3-Cl)(5-OCHF₂)—(S)CH(OAc)C(O)OH(b)

A mixture of Ph(3-Cl)(5-OCHF₂)-(R,S)CH(OH)C(O)OH (3.2 g, 12.7 mmol; seestep (vii) above) and Lipase PS “Amano” (˜2.0 g) in vinyl acetate (125mL) and MTBE (125 mL) was heated at reflux for 48 h. The reactionmixture was cooled, filtered through Celite® and the filter cake washedwith EtOAc. The filtrate was concentrated in vacuo and subjected toflash chromatography on silica gel eluting with CHCl₃:MeOH:concentratedNH₄OH (6:3:1) yielding the ammonium salts of the sub-title compounds (a)and (b). Compound (a) as a salt was dissolved in H₂O, acidified with 2NHCl and extracted with EtOAc. The organic layer was washed with brine,dried (Na₂SO₄), filtered and concentrated in vacuo to afford thesub-title compound (a) (1.2 g, 37%).

For sub-title compound (a)

¹H NMR (300 MHz, CD₃OD) δ 7.38 (s, 1H), 7.22 (s, 1H), 7.15 (s, 1H), 6.89(t, J_(H-F)=71.1 Hz, 1H), 5.17 (s, 1H)

(ix) 2,6-Difluoro-4[(methylsulfinyl)(methylthio)methyl]benzonitrile

(Methylsulfinyl)(methylthio)methane (7.26 g, 0.0584 mol) was dissolvedin 100 mL of dry THF under argon and was cooled to −78° C. Butyllithiumin hexane (16 mL 1.6M, 0.0256 mol) was added dropwise with stirring. Themixture was stirred for 15 min. Meanwhile, a solution of3,4,5-trifluorobenzonitrile (4.0 g, 0.025 mmol) in 100 mL of dry THF wascooled to −78° C. under argon and the former solution was added througha cannula to the latter solution over a period of 35 min. After 30 min,the cooling bath was removed and when the reaction had reached roomtemperature it was poured into 400 mL of water. The THF was evaporatedand the remaining aqueous layer was extracted three times with diethylether. The combined ether phase was washed with water, dried (Na₂SO₄)and evaporated. Yield: 2.0 g (30%).

¹H NMR (500 MHz, CDCl₃) δ 7.4–7.25 (m, 2H), 5.01 (s, 1H, diasteromer),4.91 (s, 1H, diasteromer), 2.88 (s, 3H, diasteromer), 2.52 (s, 3H,diasteromer), 2.49 (s, 3H, diasteromer), 2.34 (s, 3H, diasteromer), 1.72(broad, 1H)

(x) 2,6-Difluoro-4-formylbenzonitrile

2,6-Difluoro-4[(methylsulfinyl)(methylthio)methyl]benzonitrile (2.17 g,8.32 mmol; see step (ix) above) was dissolved in 90 mL of THF and 3.5 mLof concentrated sulfuric acid was added. The mixture was left at roomtemperature for 3 days and subsequently poured into 450 mL of water.Extraction three times with EtOAc followed and the combined etherealphase was washed twice with aqueous sodium bicarbonate and with brine,dried (Na₂SO₄) and evaporated. Yield: 1.36 g (98%). The position of theformyl group was established by ¹³C NMR. The signal from the fluorinatedcarbons at 162.7 ppm exhibited the expected coupling pattern with twocoupling constants in the order of 260 Hz and 6.3 Hz respectivelycorresponding to an ipso and a meta coupling from the fluorine atoms.

¹H NMR (400 MHz, CDCl₃) δ 10.35 (s, 1H), 7.33 (m, 2H)

(xi) 2,6-Difluoro-4-hydroxymethylbenzonitrile

2,6-Difluoro-4-formylbenzonitrile (1.36 g, 8.13 mmol; see step (x)above) was dissolved in 25 mL of methanol and cooled on an ice bath.Sodium borohydride (0.307 g, 8.12 mmol) was added in portions withstirring and the reaction was left for 65 min. The solvent wasevaporated and the residue was partitioned between diethyl ether andaqueous sodium bicarbonate. The ethereal layer was washed with moreaqueous sodium bicarbonate and brine, dried (Na₂SO₄) and evaporated. Thecrude product crystallised soon and could be used without furtherpurification. Yield: 1.24 g (90%).

¹H NMR (400 MHz, CDCl₃) δ 7.24 (m, 2H), 4.81 (s, 2H), 2.10 (broad, 1H)

(xii) 4-Cyano-2,6-difluorobenzyl methanesulfonate

To an ice cooled solution of 2,6-difluoro-4-hydroxymethylbenzonitrile(1.24 g, 7.32 mmol; see step (xi) above) and methanesulfonyl chloride(0.93 g, 8.1 mmol) in 60 mL of methylene chloride was addedtriethylamine (0.81 g, 8.1 mmol) with stirring. After 3 h at 0° C., themixture was washed twice with 1M HCl and once with water, dried (Na₂SO₄)and evaporated. The product could be used without further purification.Yield: 1.61 g (89%).

¹H NMR (300 MHz, CDCl₃) δ 7.29 (m, 2H), 5.33 (s, 2H), 3.07 (s, 3H)

(xiii) 4-Azidomethyl-2,6-difluorobenzonitrile

A mixture of 4-cyano-2,6-difluorobenzyl methanesulfonate (1.61 g, 6.51mmol; see step (xii) above) and sodium azide (0.72 g, 0.0111 mol) in 10mL of water and 20 mL of DMF was stirred at room temperature overnight.The resultant was subsequently poured into 200 mL of water and extractedthree times with diethyl ether. The combined ethereal phase was washedfive times with water, dried (Na₂SO₄) and evaporated. A small sample wasevaporated for NMR purposes and the product crystallised. The rest wasevaporated cautiously but not until complete dryness. Yield(theoretically 1.26 g) was assumed to be almost quantitative based onNMR and analytical HPLC.

¹H NMR (400 MHz, CDCl₃) δ 7.29 (m, 2H), 4.46 (s, 2H)

(xiv) 4-Aminomethyl-2,6-difluorobenzonitrile

This reaction was carried out according to the procedure described in J.Chem. Res. (M) (1992) 3128. To a suspension of 520 mg of 10% Pd/C (50%moisture) in 20 mL of water was added a solution of sodium borohydride(0.834 g, 0.0221 mol) in 20 mL of water. Some gas evolution resulted.4-Azidomethyl-2,6-difluorobenzonitrile (1.26 g, 6.49 mmol; see step(xiii) above) was dissolved in 50 mL of THF and added to the aqueousmixture on an ice bath over 15 min. The mixture was stirred for 4 h,whereafter 20 mL of 2M HCl was added and the mixture was filteredthrough Celite. The Celite was rinsed with more water and the combinedaqueous phase was washed with EtOAc and subsequently made alkaline with2M NaOH. Extraction three times with methylene chloride followed and thecombined organic phase was washed with water, dried (Na₂SO₄) andevaporated. Yield: 0.87 g (80%).

¹H NMR (400 MHz, CDCl₃) δ 7.20 (m, 2H), 3.96 (s, 2H), 1.51 (broad, 2H)

(xv) 2,6-Difluoro-4-tert-butoxycarbonylaminomethylbenzonitrile

A solution of 4-aminomethyl-2,6-difluorobenzonitrile (0.876 g, 5.21mmol; see step (xiv) above) was dissolved in 50 mL of THF anddi-tert-butyl dicarbonate (1.14 g, 5.22 mmol) in 10 mL of THF was added.The mixture was stirred for 3.5 h. The THF was evaporated and theresidue was partitioned between water and EtOAc. The organic layer waswashed three times with 0.5 M HCl and water, dried (Na₂SO₄) andevaporated. The product could be used without further purification.Yield: 1.38 g (99%).

¹H NMR (300 MHz, CDCl₃) δ 7.21 (m, 2H), 4.95 (broad, 1H), 4.43 (broad,2H), 1.52 (s, 9H)

(xvi) Boc-Pab(2,6-diF)(OH)

A mixture of 2,6-difluoro-4-tert-butoxycarbonylaminomethylbenzonitrile(1.38 g, 5.16 mmol; see step (xv) above), hydroxylamine hydrochloride(1.08 g, 0.0155 mol) and triethylamine (1.57 g, 0.0155 mol) in 20 mL ofethanol was stirred at room temperature for 36 h. The solvent wasevaporated and the residue was partitioned between water and methylenechloride. The organic layer was washed with water, dried (Na₂SO₄) andevaporated. The product could be used without further purification.Yield: 1.43 g (92%).

¹H NMR (500 MHz, CD₃OD) δ 7.14 (m, 2H), 4.97 (broad, 1H), 4.84 (broad,2H), 4.40 (broad, 2H), 1.43 (s, 9H)

(xvii) Boc-Pab(2,6-diF)×HOAc

This reaction was carried out according to the procedure described byJudkins et al, Synth. Comm. (1998) 4351. Boc-Pab(2,6-diF)(OH) (1.32 g,4.37 mmol; see step (xvi) above), acetic anhydride (0.477 g, 4.68 mmol)and 442 mg of 10% Pd/C (50% moisture) in 100 mL of acetic acid washydrogenated at 5 atm pressure for 3.5 h. The mixture was filteredthrough Celite, rinsed with ethanol and evaporated. The residue wasfreeze-dried from acetonitrile and water and a few drops of ethanol. Thesub-title product could be used without further purification. Yield:0.1.49 g (99%).

¹H NMR (400 MHz, CD₃OD) δ 7.45 (m, 2H), 4.34 (s, 2H), 1.90 (s, 3H), 1.40(s, 9H)

(xviii) Boc-Pab(2,6-diF)(Teoc)

To a solution of Boc-Pab(2,6-diF)×HOAc (1.56 g, 5.49 mmol; see step(xvii) above) in 100 mL of THF and 1 mL of water was added2-(trimethylsilyl)ethyl p-nitrophenyl carbonate (1.67 g, 5.89 mmol). Asolution of potassium carbonate (1.57 g, 0.0114 mol) in 20 mL of waterwas added dropwise over 5 min. The mixture was stirred overnight. TheTHF was evaporated and the residue was partitioned between water andmethylene chloride. The aqueous layer was extracted with methylenechloride and the combined organic phase was washed twice with aqueoussodium bicarbonate, dried (Na₂SO₄) and evaporated. Flash chromatographyon silica gel with heptane/EtOAc=2/1 gave 1.71 g (73%) of pure compound.

¹H NMR (400 MHz, CDCl₃) δ 7.43 (m, 2H), 4.97 (broad, 1H), 4.41 (broad,2H), 4.24 (m, 2H), 1.41 (s, 9H), 1.11 (m, 2H), 0.06 (s, 9H)

(xix) Boc-(S)Aze-Pab(2,6-diF)(Teoc)

Boc-Pab(2,6-diF)(Teoc) (1.009 g, 2.35 mmol; see step (xviii) above) wasdissolved in 50 mL of EtOAc saturated with HCl(g). The mixture was leftfor 10 min., evaporated and dissolved in 18 mL of DMF, and then cooledon an ice bath. Boc-(S)Aze-OH (0.450 g, 2.24 mmol), PyBOP (1.24 g, 2.35mmol) and lastly diisopropylethyl amine (1.158 g, 8.96 mmol) were added.The reaction mixture was stirred for 2 h and then poured into 350 mL ofwater and extracted three times with EtOAc. The combined organic phasewas washed with brine, dried (Na₂SO₄) and evaporated. Flashchromatography on silica gel with heptane:EtOAc (1:3) gave 1.097 g (96%)of the desired compound.

¹H NMR (500 MHz, CDCl₃) δ 7.46 (m, 2H), 4.65–4.5 (m, 3H), 4.23 (m, 2H),3.87 (m, 1H), 3.74 (m, 1H), 2.45–2.3 (m, 2H), 1.40 (s, 9H), 1.10 (m,2H), 0.05 (s, 9H)

(xx) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(Teoc)

Boc-(S)Aze-Pab(2,6-diF)(Teoc) (0.256 g, 0.500 mmol; see step (xix)above) was dissolved in 20 mL of EtOAc saturated with HCl(g). Themixture was left for 10 min. and evaporated and dissolved in 5 mL ofDMF. Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)OH (0.120 g, 0.475 mmol; see step(viii) above), PyBOP (0.263 g, 0.498 mmol) and lastly diisopropylethylamine (0.245 g, 1.89 mmol were added. The reaction mixture was stirredfor 2 h and then poured into 350 mL of water and extracted three timeswith EtOAc. The combined organic phase was washed with brine, dried(Na₂SO₄) and evaporated. Flash chromatography on silica gel with EtOAcgave 0.184 g (60%) of the desired sub-title compound.

¹H NMR (400 MHz, CD₃OD, mixture of rotamers) δ 7.55–7.45 (m, 2H), 7.32(m, 1H, major rotamer), 7.27 (m, 1H, minor rotamer), 7.2–7.1 (m, 2H),6.90 (t, 1H, major rotamer), 6.86 (t, 1H, minor rotamer), 5.15 (s, 1H,major rotamer), 5.12 (m, 1H, minor rotamer), 5.06 (s, 1H, minorrotamer), 4.72 (m, 1H, major rotamer), 4.6–4.45 (m, 2H), 4.30 (m, 1H,major rotamer), 4.24 (m, 2H), 4.13 (m, 1H, major rotamer), 4.04 (m, 1H,minor rotamer), 3.95 (m, 1H, minor rotamer), 2.62 (m, 1H, minorrotamer), 2.48 (m, 1H, major rotamer), 2.22 (m, 1H, major rotamer), 2.10(m, 1H, minor rotamer), 1.07 (m, 2H), 0.07 (m, 9H)

(xxi) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(Teoc) (81 mg, 0.127mmol; see step (xx) above) was dissolved in 0.5 mL of methylene chlorideand cooled on an ice bath. TFA (3 mL) was added and the reaction wasleft for 75 min. The TFA was evaporated and the residue was freeze driedfrom water and acetonitrile. The crude product was purified bypreparative RPLC with CH₃CN:0.1M NH₄OAc (35:65) to produce 39 mg (55%)of the title compound as its HOAc salt, purity: 99%.

¹H NMR (400 MHz, CD₃OD mixture of rotamers) δ 7.5–7.4 (m, 2H), 7.32 (m,1H, major rotamer), 7.28 (m, 1H, minor rotamer), 7.2–7.1 (m, 3H) 6.90(t, 1H, major rotamer), 6.86 (t, minor rotamer), 5.15 (s, 1H, majorrotamer), 5.14 (m, 1H, minor rotamer), 5.07 (s, 1H, minor rotamer), 4.72(m, 1H, major rotamer), 4.65–4.45 (m, 2H), 4.30 (m, 1H, major rotamer),4.16 (m, 1H, major rotamer), 4.03 (m, 1H, minor rotamer), 3.95 (m, 1H,minor rotamer), 2.63 (m;, 1H, minor rotamer), 2.48 (m, 1H, majorrotamer), 2.21 (m, 1H, major rotamer), 2.07 (m, 1H, minor rotamer), 1.89(s, 3H) ¹³C-NMR (75 MHz; CD₃OD): (carbonyl and/or amidine carbons,mixture of rotamers) δ 171.9, 171.2, 165.0, 162.8, 160.4; APCI-MS:(M+1)=503/505 m/z.

EXAMPLE 2 Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe)

(i) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe,Teoc)

A mixture of Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(Teoc)(64 mg, 0.099 mmol; see Example 1(xx) above) and O-methyl hydroxylaminehydrochloride (50 mg, 0.60 mmol) in 4 mL of acetonitrile was heated at70° C. for 3 h. The solvent was evaporated and the residue waspartitioned between water and EtOAc. The aqueous layer was extractedtwice with EtOAc and the combined organic phase was washed with water,dried (Na₂SO₄) and evaporated. The product could be used without furtherpurification. Yield: 58 mg (87%).

¹H NMR (400 MHz, CDCl₃) δ 7.90 (bt, 1H), 7.46 (m, 1H), 7.25–6.95 (m,5H), 6.51, t, 1H), 4.88 (s, 1H), 4.83 (m, 1H), 4.6–4.5 (m, 2H), 4.4–3.9(m, 4H), 3.95 (s, 3H), 3.63 (m, 1H), 2.67 (m, 1H), 2.38 (m, 1H), 1.87(broad, 1H), 0.98 (m, 2H), 0.01, s, 9H)

(ii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OMe,Teoc) (58 mg,0.086 mmol; see step (i) above) was dissolved in 3 mL of TFA, cooled onan ice bath and allowed to react for 2 h. The TFA was evaporated and theresidue dissolved in EtOAc. The organic layer was washed twice withaqueous sodium carbonate and water, dried (Na₂SO₄) and evaporated. Theresidue was freeze-dried from water and acetonitrile to give 42 mg (92%)of the title compound. Purity: 94%.

¹H NMR (300 MHz, CDCl₃) δ 7.95 (bt, 1H), 7.2–7.1 (m, 4H), 6.99 (m, 1H),6.52 (t, 1H), 4.88 (s, 1H), 4.85–4.75 (m, 3H), 4.6–4.45 (m, 2H), 4.29(broad, 1H), 4.09 (m, 1H), 3.89 (s, 3H), 3.69 (m, 1H), 2.64 (m, 1H),2.38 (m, 1H), 1.85 (broad, 1H) ¹³C-NMR (100 MHz; CDCl₃): (carbonyland/or amidine carbons) δ 172.1, 169.8, 151.9 APCI-MS: (M+1) 533/535 m/z

EXAMPLE 3 Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH)

(i) Boc-(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)

Boc-(S)Aze-OH (1.14 g, 5.6 mmol) was dissolved in 45 mL of DMF.4-Aminomethyl-2,6-difluorobenzonitrile (1.00 g, 5.95 mol, see Example1(xiv) above), PyBOP (3.10 g, 5.95 mmol) and DIPEA (3.95 mL, 22.7 mmol)were added and the solution was stirred at room temperature for 2 h. Thesolvent was evaporated and the residue was partitioned between H₂O andEtOAc (75 mL each). The aqueous phase was extracted with 2×50 mL EtOAcand the combined organic phase was washed with brine and dried overNa₂SO₄. Flash chromatography (SiO₂, EtOAc/heptane (3/1)) yielded thesub-title compound (1.52 g, 77%) as an oil which crystallized in therefrigerator.

¹H-NMR (400 MHz; CD₃OD): δ 7.19 (m, 2H), 4.65–4.5 (m, 3H), 3.86 (m, 1H),3.73 (m, 1H), 2.45–2.3 (m, 2H), 1.39 (s, 9H)

(ii) H-(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)×HCl

Boc-(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN) (0.707 g, 2.01 mmol, see step (i)above) was dissolved in 60 mL of EtOAc saturated with HCl(g). Afterstirring at room temperature for 15 minutes, the solvent was evaporated.The residue was dissolved in CH₃CN/H₂O (1/1) and was freeze-dried togive the sub-title compound (0.567 g, 98%) as an off-white amorphouspowder.

¹H-NMR (400 MHz; CD₃OD): δ 7.49 (m, 2H), 4.99 (m, 1H), 4.58 (m, 2H),4.12 (m, 1H), 3.94 (m, 1H), 2.80 (m, 1H), 2.47 (m, 1H) MS (m/z) 252.0(M+1)⁺

(iii) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-NHCH₂₋Ph(2,6-diF, 4-CN)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)OH (0.40 g, 1.42 mmol, see Example1(viii) above) was dissolved in 10 mL of DMF andH-(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN)×HCl (0.43 g, 1.50 mmol, see step (ii)above) and PyBOP (0.779 g, 1.50 mmol) were added, followed by DIPEA (1.0mL, 5.7 mmol). After stirring at room temperature for 2 h, the solventwas is evaporated. The residue was partitioned between H₂O (200 mL) andEtOAc (75 mL). The aqueous phase was extracted with 2×75 mL EtOAc andthe combined organic phase was washed with brine and dried over Na₂SO₄.Flash chromatography (SiO₂, EtOAc/heptane (4/1)) yielded the sub-titlecompound (0.56 g, 81%) as an oil.

¹H-NMR (400 MHz; CD₃OD) rotamers: δ 7.43 (m, 2H), 7.31 (m, 1H, majorrotamer), 7.26 (m, 1H, minor rotamer), 7.2–7.1 (m, 2H), 6.90 (t, 1H,major rotamer), 6.86 (t, 1H, minor rotamer), 5.14 (s, 1H, majorrotamer), 5.11 (m, 1H, minor rotamer), 5.04 (s, 1H, minor rotamer), 4.71(m, 1H, major rotamner), 4.6–4.45 (m, 2H), 4.30 (m, 1H, major rotamer),4.2-3.9 (m, 1H; and 1H, minor rotamer), 2.62 (m, 1H, minor rotamer),2.48 (m, 1H, major rotamer), 2.21 (m, 1H, major rotamer), 2.09 (m, 1H,minor rotamer) ¹³C-NMR (100 MHz; CD₃OD): (carbonyl carbons) δ 171.9,171.8; MS (m/z) 484.0, 485.9 (M−1)⁻, 486.0, 487.9 (M+1)⁺

(iv) Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH)

Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-NHCH₂-Ph(2,6-diF, 4-CN) (0.555 g,1.14 mmol, from step (iii) above) was dissolved in 10 mL of EtOH. (95%).To this solution was added hydroxylamine hydrochloride (0.238 g, 3.42mmol) and Et₃N (0.48 mL, 3.44 mmol). After stirring at room temperaturefor 14 h, the solvent was removed and the residue was dissolved inEtOAc. The organic phase was washed with brine and H₂O and was driedover Na₂SO₄. The crude product was purified by preparative to RPLC withCH₃CN:0.1 M NH₄OAc as eluent, yielding the title compound as anamorphous powder (0.429 g, 72%) after freeze-drying.

¹H-NMR (400 MHz; CD₃OD) rotamers: δ 7.35–7.1 (m, 5H), 6.90 (t, 1H, majorrotamer), 6.85 (t, 1H, minor rotamer), 5.15 (s, 1H, major rotamer), 5.12(m, 1H, minor rotamer), 5.08 (s, 1H, minor rotamer), 4.72 (m, 1H, majorrotamer), 4.6–4.4 (m, 2H), 4.30 (m, 1H, major rotamer), 4.12 (m, 1H,major rotamer), 4.04 (m, 1H, minor rotamer), 3.94 (m, 1H, minorrotamer), 2.62 (m, 1H, minor rotamer), 2.48 (m, 1H, major rotamer), 2.22(m, 1H, major rotamer), 2.10 (m, 1H, minor rotamer) ¹³C-NMR (100 MHz;CD₃OD): (carbonyl and amidine carbons, rotamers) δ 172.4, 171.9, 171.0,152.3, 151.5 MS (m/z) 517.1, 519.0 (M−1)⁻, 519.1, 521.0 (M+1)⁺

EXAMPLE 4

The title compound of Example 1 was tested in Test A above and was foundto exhibit an IC₅₀TT value of less than 0.02 μM.

EXAMPLE 5

The title compound of Example 1 was tested in Test D above and was foundto exhibit an IC₅₀ APTT value of less than 1 μM.

EXAMPLE 6

The title compound of Example 2 was tested in Test E above and was foundto exhibit oral and/or parenteral bioavailability in the rat as thecorresponding active inhibitor (free amidine).

EXAMPLE 7

The title compound of Example 2 was tested in Test G above and was foundto be converted to the corresponding active inhibitor (free amidine) inliver microsomes from humans and from rats.

ABBREVIATIONS

-   Ac=acetyl-   APCI=atmospheric pressure chemical ionisation (in relation to MS)-   API=atmospheric pressure ionisation (in relation to MS)-   aq.=aqueous-   AUC=area under the curve-   Aze=azetidine-2-carboxylate-   AzeOH=azetidine-2-carboxylic acid-   Boc=tert-butyloxycarbonyl-   BSA=bovine serum albumin-   CI=chemical ionisation (in relation to MS)-   d=day(s)-   DCC=dicyclohexyl carbodiimide-   DIBAL-H=di-isobutylaluminium hydride-   DIPEA=diisopropylethylamine-   DMAP=4-(N,N-dimethyl amino)pyridine-   DMF=dimethylformamide-   DMSO=dimethylsulfoxide-   DVT=deep vein thrombosis-   EDC=1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride-   Et=ethyl-   ether=diethyl ether-   EtOAc=ethyl acetate-   EtOH=ethanol-   Et₂O=diethyl ether-   h=hour(s)-   HATU=O-(azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HBTU=[N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium    hexafluorophosphate]-   HCl=hydrochloric acid, hydrogen chloride gas or hydrochloride salt    (depending on context)-   Hex=hexanes-   HOAc=acetic acid or acetic acid salt-   HPLC=high performance liquid chromatography-   LC=liquid chromatography-   Me=methyl-   MeOH=methanol-   min=minute(s)-   MS=mass spectroscopy-   MTBE=methyl tert-butyl ether-   NADH=nicotinamide adenine dinucleotide, reduced form-   NADPH=nicotinamide adenine dinucleotide phosphate, reduced form-   NIH=National Institute of Health (US)-   NIHU=National Institute of Health units-   NMR=nuclear magnetic resonance-   OAc=acetate-   Pab=para-amidinobenzylamino-   H-Pab=para-amidinobenzylamine-   Ph=phenyl-   PyBOP=(benzotriazol-1-yloxy)tripyrrolidinophosphonium    hexafluorophosphate-   QF=tetrabutylammonium fluoride-   RPLC=reverse phase high performance liquid chromatography-   rt/RT=room temperature-   SOPs=standard operating procedures-   TBTU=[N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium    tetrafluoroborate]-   TEA=triethylamine-   Teoc=2-(trimethylsilyl)ethoxycarbonyl-   TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxy free radical-   TFA=trifluoroacetic acid-   THF=tetrahydrofuran-   TLC=thin layer chromatography-   UV=ultraviolet

Prefixes n, s, i and t have their usual meanings: normal, secondary, isoand tertiary. The prefix c means cyclo.

1. A compound Ph(3-Cl)(5-OCHF₂)—(R)CH(OH)C(O)—(S)Aze-Pab(2,6-diF)(OH) ora pharmaceutically-acceptable salt thereof.
 2. A pharmaceuticalformulation including a compound as defined in claim 1, or apharmaceutically acceptable salt thereof, in admixture with apharmaceutically acceptable adjuvant, diluent or carrier.
 3. A method oftreatment of thrombosis, which method comprises administration of atherapeutically effective amount of a compound as defined in claim 1, ora pharmaceutically acceptable salt thereof, to a person suffering from,or susceptible to, such a condition.
 4. A method of treatment ofhypercoagulability in blood and/or tissues, which method comprisesadministration of a therapeutically effective amount of a compound asdefined in claim 1, or a pharmaceutically acceptable salt thereof, to aperson suffering from, or susceptible to, such a condition.
 5. A processfor the preparation of a compound of claim 1: or apharmaceutically-acceptable salt thereof, which comprises: (a) reactionof a compound of formula II:

 with a compound of formula XIV,

 wherein R¹ represents OH; (b) reaction of a compound of formula IV:

 with a compound of formula XV,

 wherein R¹ represents OH; (c) reaction of a compound of formula XVI,

 with hydroxylamine; (d) reaction of a compound of formula XVII,

 wherein R^(a) represents —CH₂CH₂—Si(CH₃)₃ or benzyl, or a tautomerthereof, with hydroxylamine, or an acid addition salt thereof, followedby removal of the —C(O)OR^(a) group; (e) reaction of a compound offormula XVII, as defined above, in which R^(a) represents benzyl, withhydroxylamine, or an acid addition salt thereof.